Our objectives are to determine the genetic basis for virulence and adaptation to cell culture of HAV in order to determine if it is possible to develop a strain of HAV suitable for use as an attenuated vaccine. We established a clonal line of liver cells which is unique in that it supports the replication of clinical isolates (virulent virus) of HAV. However, virus passaged only four times in these cells became attenuated for tamarins. The basis for this attenuation is being analyzed since these cells appear to apply different selective pressures to virus replication than do other cells we have used. We are also using microarray technology to determine how this clonal cell line differs from the parent cell line. In all cell culture systems studied thus far (except for our unique liver cell line) viral replication requires a specific amino acid change in one of the nonstructural proteins not involved in virulence. This site was mutagenized and the effect of different amino acid substitutions on replication in vitro and in vivo were determined.